Performance Of Assay Research Articles

Establishment and Validation of Fecal Secretory Immunoglobulin A Measurement for Intestinal Mucosal Health Assessment in Wild Lemurs.

The measurement of biomarkers in blood and excreta can enable immune status assessment and provide prognostic information on individual health outcomes. In this respect, the fecal measurement of secretory immunoglobulin A (sIgA), the primary mammalian antibody for mucosal defense, has recently received increased interest in a few anthropoid primates, but a fecal sIgA assay for use in strepsirrhine primates has not yet been reported. Here, we develop and analytically validate a cost-effective in-house sandwich enzyme immunoassay for the extraction and measurement of sIgA in feces of redfronted lemurs (Eulemur rufifrons). We also tested a simple method for sIgA extraction that can be used under remote field conditions and undertook experiments to assess the robustness of sIgA concentrations to variation in processing and storage conditions of fecal extracts. Our analytical validation revealed that the assay recognizes immunoreactive sIgA in redfronted lemur feces, that sIgA can be measured accurately with no potential interference from the fecal matrix, and that assay reagents and performance are highly stable over time. The field-friendly extraction procedure produced sIgA results strongly correlated with those generated by a standard laboratory extraction method. Short-term storage at room temperature resulted in a slight decline in sIgA concentrations, whereas freezing extracts at -20°C kept sIgA levels stable for at least 3 months. Longer-term storage of >5 months, however, led to a significant decline of sIgA concentrations. Multiple freeze-thaw cycles did not affect sIgA levels. This study, therefore, provides the basis for measuring fecal sIgA in lemurs and possibly other strepsirrhines. When samples are processed properly and stored frozen, and when sIgA analysis can be performed within 3 months upon sample collection, fecal sIgA measurements can become a valuable tool for monitoring aspects of immunity and health in both zoo-housed and wild-living lemurs.

Detection and staging of Alzheimer's disease by plasma pTau217 on a high throughput immunoassay platform

Plasma phospho-tau 217 (pTau217) assays can accurately detect Alzheimer's disease (AD) pathology, but clinical application is limited by the need for specialised equipment. This study tests the performance of a plasma pTau217 assay performed on the Lumipulse-G® platform, that is in widespread clinical use, for selecting patients for therapy based on β-amyloid (Aβ) status and tau staging. Participants included 388 individuals with 18F-NAV4694 Aβ-PET and 18F-MK6240 tau-PET. Association of pTau217 with PET was examined using Spearman's correlation. Discriminative performance for Aβ and tau PET status as well as tau staging was assessed using Receiver Operating Characteristic analysis. Plasma pTau217 had a high correlation with both Aβ Centiloid (r=0.76) and tau SUVRmeta-temporal (r=0.78). Area under curve (AUC) was 0.93 for Aβ- vs Aβ+ and 0.94 for tau- vs tau+. Applying one threshold (Youden's index), pTau217 was 87% accurate in classification of participants to Aβ- vs Aβ+. Applying two thresholds to classify participants into Low, Indeterminate, and High zones, 17.8% had Indeterminate results and among Low/High zone participants, 92% were correctly classified as Aβ- or Aβ+. The assay accurately discriminated moderate/high neocortical tau from no tau or tau limited to mesial-temporal lobe (AUC 0.97) and high neocortical tau from all others (AUC 0.94). Plasma pTau217, measured by the widely-available, fully-automated Lumipulse®, was a strong predictor of both Aβ and tau PET status and demonstrated strong predictive power in identifying individuals likely to benefit the most from anti-Aβ treatments. NHMRC grants 1132604, 1140853, 1152623 and AbbVie.

Development of an optimized SEC method for characterization of genome DNA leakage from adeno-associated virus products.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.

Performance of a Global Functional Assay Based on Interferon-γ Release to Predict Infectious Complications and Cancer After Kidney Transplantation

The QuantiFERON-Monitor assay (QTF-Monitor) is intended to assess innate and adaptive immune responses by quantifying interferon (IFN)-γ release upon whole blood stimulation with a TLR7/8 agonist and an anti-CD3 antibody. We performed the QTF-Monitor in 126 kidney transplant recipients (KTRs) at different points during the first 6 post-transplant months. The primary outcome was overall infection, whereas secondary outcomes included bacterial infection, opportunistic infection and de novo cancer. The association between IFN-γ production and outcomes was analyzed as “low” immune responses (<15 IU/mL) and as a continuous variable to explore alternative thresholds. There were no significant differences in the occurrence of overall infection according to the QTF-Monitor at any monitoring point. Regarding secondary outcomes, KTRs with a low response at week 2 experienced a higher incidence of bacterial infection (50.8% versus 24.4%; P-value = 0.006). Low response at month 1 was also associated with opportunistic infection (31.6% versus 14.3%; P-value = 0.033). The discriminative capacity of IFN-γ levels was poor (areas under the ROC curve: 0.677 and 0.659, respectively). No differences were observed for the remaining points or post-transplant cancer. In conclusion, the QTF-Monitor may have a role to predict bacterial and opportunistic infection in KTRs when performed early after transplantation.

Comparison of 2 Sets of Immunoassays Used in Modified 2-Tiered Testing Algorithms for the Diagnosis of Lyme Disease.

Since 2019, modified 2-tiered testing (MTTT) algorithms have been available for the diagnosis of Lyme disease. MTTTs replaced the standard algorithms that utilized enzyme immunoassays and immunoblots with sequential enzyme immunoassays that detect different antigens. We compared the performance of serological assays from ZEUS Scientific Inc. and DiaSorin Inc. that are used for the diagnosis of Lyme disease. Serological results were compared with clinical information gathered by chart review. Percent positive agreement (PPA) and percent negative agreement (PNA) for total immunoglobulin G (IgG)/immunoglogulin M (IgM) (n = 120) were 64% (95% confidence interval 54% to 73%) and 100% (87% to 100%), respectively. PPA and PNA for IgG (n = 93) were 91% (80% to 97%) and 66% (52% to 78%), respectively. PPA and PNA for IgM (n = 93) were 75% (62% to 85%) and 95% (82% to 99%), respectively. Fewer positive total IgG/IgM results confirmed positive for either IgG or IgM for ZEUS compared to DiaSorin. Overall MTTT algorithm interpretation was concordant in 58% (55/95) of samples, and concordance improved when the results were limited to IgM in patients with symptom duration <30 days. Treatment with antibiotics was most strongly associated with IgM positivity. This analysis highlights differences in the performance characteristics between commercially available diagnostic assays for Lyme disease. Our data suggest that the DiaSorin assays would result in fewer positive total IgG/IgM tests, decreasing the required number of confirmatory IgG and IgM tests. This would potentially lead to fewer patients treated with antibiotics.

Hybrid Quadrupole Mass Filter-Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics.

Targeted mass spectrometry (MS) methods are powerful tools for the selective and sensitive analysis of peptides identified in global discovery experiments. Selected reaction monitoring (SRM) is the most widely accepted clinical MS method due to its reliability and performance. However, SRM and parallel reaction monitoring (PRM) are limited in throughput and are typically used for assays with around 100 targets or fewer. Here we introduce a new MS platform featuring a quadrupole mass filter, collision cell, and linear ion trap architecture, capable of targeting 5000-8000 peptides per hour. This high multiplexing capability is facilitated by acquisition rates of 70-100 Hz and real-time chromatogram alignment. We present a Skyline external software tool for building targeted methods based on data-independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. Our platform demonstrates ∼10× lower limits of quantitation (LOQs) than traditional SRM on a triple quadrupole instrument for highly multiplexed assays, due to parallel product ion accumulation. Finally, we explore how analytical figures of merit vary with method duration and the number of analytes, providing insights into optimizing assay performance.

Imprecision and real-time clinical performance of a whole blood high sensitivity point of care troponin i: ready for prime time?

Abstract Introduction High sensitivity troponin (hs-cTn) measurement is central to rapid diagnostic algorithms in acute coronary syndrome (ACS). Point of care (POC) testing reduces turnaround time when compared to central lab (CL) testing. Currently there is very little data on the performance of POC hs-cTn assays in a clinical environment with non-expert operators. Method In a nested sub-study of a randomised controlled trial (RCT), patients with suspected ACS were randomised to either the ESC 0/1 or 0/3 hour algorithm. They underwent whole blood (WB) sampling for Siemens Atellica VTLi hs-cTnI POC (VTLi POC), CL hs-cTnI and CL hs-cTnT assays. This was for both initial and repeat samples. Assay imprecision of the VTLi POC assay was assessed by 2 runs of 10 tests with whole blood at 9 different troponin levels. 4 devices were used with samples stored at room temperature.Final diagnosis was adjudicated based on all relevant clinical and imaging data together with CL hs-cTnT as the diagnostic biomarker in clinical use (10% coefficient of variation (CV) 3-5 ng/L, 99th percentile 14 ng/L) and using the 4th universal definition of myocardial infarction (MI). Clinical performance was assessed using Receiver Operator Characteristics (ROC) curves. Results 2144 patients consented and had WB VTLi POC, CL hs-cTnI and CL hs-cTnT available. The index adjudicated type 1 MI rate was 6.1%. Assay imprecision of the VTLi POC assay demonstrated a 10% Coefficient of Variation (CV) at 9.8ng/l. This was &amp;gt;50% lower than the 99th centile of 23ng/l (average for males and females) (Figure 1). Clinical performance of the VTLi POC assay was acceptable and equivalent to CL hs-cTnT: Area Under Curve (AUC) 0.92 (0.89-0.96), 0.98 (0.96-0.99) and 0.93 (0.87-0.98) at presentation (Figure 2), 1 and 3 hours respectively. However, clinical performance was inferior to CL hs-cTnI: AUC 0.96 (0.95-0.98) and 0.96 (0.93-0.98) at presentation (p=0.0007) and 3 hours (p&amp;lt;0.0001) respectively. Conclusion The Siemens Altellica VTLi hs-cTnI POC assay has imprecision levels consistent with a high sensitivity troponin assay. It has acceptable clinical performance though inferior to the equivalent CL hs-cTnI. Further validation of this assay, particularly with optimised single sample rule-out, could facilitate its use in routine clinical practice.

A fluorescent sensor for rapid and quantitative aquatic bacteria detection based on bacterial reactive oxygen species using Ag@carbon dots composites.

A novel fluorescent sensor based on silver nanoparticle-carbon dot composites (Ag@CDs) has been developed for the rapid and quantitative detection of aquatic bacteria. The sensor operates on the principle of plasmon-enhanced resonance energy transfer, where the fluorescence of CDs is quenched by Ag nanoparticles and restored upon bacterial interaction due to the generation of reactive oxygen species. The Ag@CDs exhibit a linear response to bacterial concentration over the range 7 × 104 ~ 4 × 107 CFU·mL-1, with a low detection limit of 4 × 104 CFU·mL-1. The fluorescence recovery is rapid, reaching maximum intensity within 5 min. The method demonstrates high selectivity, with minimal interference from common ions and compounds found in municipal and industrial wastewater. The Ag@CDs-based 96-well plate assay for quantitative measurement of bacteria was developed. The assay's performance was further validated through the analysis of real water samples, showing a recovery of 94.0 ~ 102% for domestic wastewater and 97.6 ~ 106% for industrial wastewater. Also, Ag@CDs-based test strips assay for semi-quantitation were developed for rapid in-field aquatic bacteria detection. Ag@CDs can be conveniently integrated into 96-well plates and test strips, providing rapid on-site aquatic bacteria testing.

A Closer Look at the Newer GP1bM Assay Sheds Light on Unusual Findings

Abstract Von Willibrand disease (VWD) is the most common inherited bleeding disorder. The 2021 Guidelines on VWD diagnosis recommend newer assays measuring the platelet-binding activity of von Willebrand factor (VWF), such as VWF: GPIbM and VWF: GPIbR, over the traditional ristocetin cofactor assay (VWF: RCo). Among these newer assays, the GP1bM INNOVANCE VWF Ac assay obtained de novo FDA approval in October 2022. Its ristocetin-independent activity distinguishes it from other assays. Our laboratory conducted a comprehensive validation on the Stago StaR-Max platform, utilizing Siemens Standard Plasma for calibration. The VWF activity measured by the GP1bM assay was compared with the existing VWF: Ab assay (IL, HemosIL® von Willebrand Factor Activity). The validation studies revealed a good correlation between the two assays. Since implementing the GP1bM assay in October 2023, our attention has been drawn to at least four cases exhibiting unexpectedly high activity levels compared to VWF antigen levels. On average, the activity-to-antigen ratio was 1.9. Notably, all these cases showed the presence of all multimers (low, intermediate, and high). Despite extensive investigation, encompassing considerations like heterophile antibody interference using scantibodies, retesting with different lot numbers of Innovance VWF Ac reagent, and rerunning tests at an outside laboratory, the cause of this anomaly remains elusive. A notable variable lies in the platform disparity between Siemens and Stago. Given the recent introduction of this assay, it has yet to gain widespread adoption in laboratories, resulting in a limited understanding of its practical variations. While recent studies affirm the overall superior performance of newer assays compared to the ristocetin cofactor assay, intricate details of each assay remain less explored. The novelty of the GP1bM assay and its limited usage in laboratories underscore the potential emergence of stable outliers as it becomes more widely adopted. The peculiar cases in our study pose an enigma as to why the reported activity is approximately double the antigen level. As laboratories increasingly embrace and integrate the GP1bM assay, a more complete understanding of its potential variations is expected to unfold. This may elucidate the mysteries surrounding these peculiar cases and ensure the assay’s robust interpretation in clinical settings.

Exploring nanopore direct sequencing performance of forensic STRs, SNPs, InDels, and DNA methylation markers in a single assay

IntroductionThe field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow. Methods and resultsIn our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90–92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson’s correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath’s clock. Additional age-associated markers exhibited Pearson’s correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21–72, showing 100 % concordance with reference data. DiscussionOur exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases.

Use of T2Bacteria Panel in Pediatric Hematopoietic Stem Cell Transplant Patients: A Single Center Experience

Abstract Background Bloodstream infection is a common complication following hematopoietic cell transplant (HCT), occurring in 10-40% of patients (1,2). Rapid identification of causative bacteria is imperative to provide proper empiric therapy and ensure optimal patient outcomes. The T2Bacteria Panel detects five bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli) using T2 magnetic resonance (T2MR, T2Biosystems, Lexington MA, USA) directly from blood specimens. Previous studies have reported per-patient sensitivity, specificity, and negative predictive value (NPV) of 84-90%, 85.9-90%, and 99.7%, respectively, as well as 100% positive agreement and 98.4% negative agreement when compared to blood culture (3-5). To date, there have been no studies on the use of this test in pediatric HCT recipients. Methods We reviewed all pediatric HCT patients at St. Jude Children’s Research Hospital who had a T2Bacteria Panel performed for fever and suspected infection between January 2019 and September 2022. A total of 706 T2Bacteria Panel results and concurrent blood culture results from 251 unique patients were analyzed to determine assay performance for detection of the 5 targeted pathogens. Results T2Bacteria PCR and blood culture showed concordant results in 97% of cases. Overall, our study revealed 65% positive percent agreement with blood culture and 95% negative percent agreement for bacteremia caused by any of the target pathogens. PPV, NPV, sensitivity and specificity for each organism detected by the panel, and the panel overall, are presented in Table 1. Positive predictive value ranged from 28-73%. Negative predictive value was high for all organisms (98-100%). Sensitivity was highest for Pseudomonas aeruginosa (88%), and lowest for Escherichia coli (44%). Specificity was high for all organisms (97-99%). Conclusion In pediatric HSCT patients, the T2Bacteria Panel was most useful for rapidly ruling out the presence of target pathogens, with high negative percent agreement, NPV, and specificity. Positive percent agreement was lower than noted in previous studies. Positive predictive value and sensitivity varied per pathogen.

High-Throughput Hybridization Assay as First-Line Diagnostic Test for Sarcomas: Clinical Assessment in a Tertiary Referral Center.

Sarcomas are rare and highly heterogeneous mesenchymal tumors with deceptive morphologic features that pose a challenge for precise diagnostics. Chromosomal rearrangements generating pathognomonic gene fusions are useful diagnostic markers, traditionally tested using single-plex standard of care assays with limited diagnostic yield. NanoString nCounter technology has emerged as a robust solution with multiplexing capabilities. To optimize NanoString effective coverage of specific entities and conduct a validation study to support its clinical implementation. We reconfigured a NanoString's codeset by including a set of probes for detecting gene fusion variants of solitary fibrous tumors, low-grade fibromyxoid sarcomas/sclerosing epithelioid fibrosarcomas, and undifferentiated small round cell sarcomas, totaling 188 probes. A technical validation study was conducted with 96 retrospective samples. Additionally, 76 prospective samples were evaluated to assess the assay's clinical performance. Both technical and clinical validation studies showed that NanoString's codeset reached >88% sensitivity and 100% specificity, compared with standard of care methods, and superior diagnostic yield as a first-line test. Our design enabled the detection of almost all fusion variants of NGFI-A binding protein 2 (NAB2) with signal transducer and activator of transcription 6 (STAT6) in solitary fibrous tumors, as well as cAMP responsive element binding protein 3 like 1/2 (CREB3L1/2) rearrangements in all low-grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma cases. Identification of specific gene fusions of undifferentiated small round cell sarcoma was also improved, but additional strategies are necessary to attain full coverage. The NanoString platform demonstrated good sensitivity, specificity, and superior diagnostic yield. It is a cost-effective assay with rapid turnaround time, low sample consumption, streamlined analysis, and easy customization. Therefore, it is a promising alternative first-line diagnostic tool for routine sarcoma testing.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

Enhancement of loop-mediated isothermal amplification (LAMP) with guanidine hydrochloride for the detection of Streptococcus equi subspecies equi (Strangles).

Streptococcus equi subspecies equi, commonly referred to as "strangles", poses a significant biosecurity challenge across equine farms worldwide. The continuous prevalence and highly transmissibility of strangles necessitates a rapid and accurate diagnostic procedure. However, current "gold-standard" techniques, such as cultures and quantitative polymerase chain reaction (qPCR), are unreliable or inaccessible, and require lengthy periods between sample collection and results. Moreover, the lack of a standardized detection protocol can lead to variations in results. This study aimed to develop a reproducible and field-deployable diagnostic assay to detect strangles in real-time. Utilising the rapid technique loop-mediated isothermal amplification (LAMP), we developed an assay targeting a conserved region of the S. equi-specific M gene (SeM). Additionally, we optimised our assay with guanidine hydrochloride (GuHCl) to enhance the assay's performance and detection capabilities. The Str-LAMP was able to detect S. equi within 13 minutes and 20 seconds for both synthetic DNA and clinical isolates, with a limit of detection (LOD) of 53 copies/µl. Our assay demonstrated high repeatability with the inter-coefficient of variation ranging from 0.17% to 3.93%. Furthermore, the clinical sensitivity and specificity was calculated at 91.3% and 93.3%, respectively, with a correct classification rate of 91.8%. The implementation of this newly developed strangles assay can be employed as an efficient aid for in-field surveillance programs. The assay's reproducibility can allow for equine managers to undertake routine self-surveillance on their properties, without the requirement of specialised training. The Str-LAMP assay has the potential to be a valuable tool to help mitigate potential strangles outbreaks.

The Utility of an Human Papillomavirus Genotype Assay for Cancer Screening in Self-Collected Urine and Vaginal Samples from Japanese Women.

The high incidence of invasive cervical cancer among those who have not undergone cancer screening is a serious problem. This study aimed to investigate the utility of human papillomavirus (HPV) test results from self-collected urine and vaginal samples as screening tools. The study was conducted in two steps. First, the appropriate storage container, temperature, and time until urine HPV assay performance were verified. Second, the results of spot urine testing under those conditions and of gynecologist-collected cervical and self-collected vaginal samples were compared to verify the feasibility of using the BD Onclarity® HPV assay for individuals with abnormal cervical cytology. The participants were 121 women with abnormal cervical cytology. Self-collected urine and vaginal samples, along with gynecologist-collected cervical samples, were tested for HPV using the BD Onclarity® HPV assay. The optimal conditions for urine sample storage were identified by comparing the HPV detection rates under various conditions. Urine stored in a BD Probe Tec™ (QxUPT) for less than 72 h at room temperature was found to have the highest HPV positivity rate. Under these conditions, the detection rates of HPV in urine, cervical, and vaginal samples were examined. HPV type 16 was detected in 41.7% of the cervical samples, type 18 in 10%, and types 31 and 52 in 12.6% each. The concordance rate for HPV testing between clinician-collected cervical and urine samples was 63.9% (kappa: 0.34; 95% CI: 0.21-0.47), and that between clinician-collected cervical and self-collected vaginal samples was 77.8% (kappa: 0.68; 95% CI: 0.53-0.83), indicating good concordance. In a population with an HPV-related lesion/tumor prevalence of approximately 70%, the sensitivity of HPV testing was 82.7% for the cervix, 46.4% for urine, and 75.7% for vaginal samples. The primary limitation is the lower detection rate of HPV in spot urine samples than in other sample types, indicating room for methodological improvement. The study's findings are based on a specific population, which may limit generalizability. We investigated the optimal self-collected urine-to-testing time and temperature. Self-collected vaginal and urine HPV tests show moderate-high concordance with clinician-collected cervical HPV tests, suggesting their potential utility for women who do not undergo regular cancer screening. However, the sensitivity was not high in spot urine. Therefore, further large-scale studies are needed to verify these findings and optimize testing methods to encourage broader participation in cancer screening programs.

An electrochemical proximity assay (ECPA) for antibody detection incorporating flexible spacers for improved performance.

A clever approach for biosensing is to leverage the concept of the proximity effect, where analyte binding to probes can be coupled to a second, controlled binding event such as short DNA strands. This analyte-dependent effect has been exploited in various sensors with optical or electrochemical readouts. Electrochemical proximity assays (ECPA) are more amenable to miniaturization and adaptation to the point-of-care, yet ECPA has been generally targeted toward protein sensing with antibody-oligonucleotide probes. Antibodies themselves are also important as biomarkers, since they are produced in bodily fluids in response to various diseases or infections, often in low amounts. In this work, by using antigen-DNA conjugates, we targeted an ECPA method for antibody sensing and showed that the assay performance can be greatly enhanced using flexible spacers in the DNA conjugates. After adding flexible polyethylene glycol (PEG) spacers at two distinct positions, the spacers ultimately increased the antibody-dependent current by a factor of 4.0 without significant background increases, similar to our recent work using thermofluorimetric analysis (TFA). The optimized ECPA was applied to anti-digoxigenin antibody quantification at concentrations ranging over two orders of magnitude, from the limit of detection of 300pM up to 50nM. The assay was functional in 90% human serum, where increased ionic strength was used to counteract double-layer repulsion effects at the electrode. This flexible-probe ECPA methodology should be useful for sensing other antibodies in the future with high sensitivity, and the mechanism for signal improvement with probe flexibility may be applicable to other DNA-based electrochemical sensor platforms.

B-113 Beckman Coulter DxI 9000 immunoassay analyzer assay performance meets new CLIA 2024 performance goals with highest proportion of Six Sigma performance amongst all major manufacturers

Abstract Background The Clinical Laboratory Improvement Act (CLIA) proficiency testing (PT) criteria will be updated in July 2024 with some goals tightening by up to 40%. It is assumed, but not known, that all assays and instruments will be able to achieve these goals. Using these new CLIA 2024 goals, estimates of current instrument group performance from an international proficiency testing (PT) survey have shown significant differences in quality, calling into question which assays can achieve the new CLIA goals. Currently, the highest performing diagnostic instrument achieves only 35.2% 6-Sigma, while the majority of instruments have less than 15% 6-Sigma performance indicating a higher probability of future PT failures. Methods Eighteen assays that will be governed by new CLIA 2024 goals were assessed for Sigma metric performance on the DxI 9000 immunoassay analyzer: Alpha Fetoprotein (AFP), Beta hCG, Carcinoembryonic antigen (CEA), Cortisol, Creatine Kinase MB (CK-MB), Estradiol, Ferritin, Folate, Follicle-Stimulating Hormone (FSH), Prolactin, Para-thyroid hormone (PTH), Total Prostate specific antigen (Total PSA), Testosterone, Thyroid Stimulating Hormone (TSH), Total Triiodothyronine (TT3),high sensitivity Troponin I, Total Thyroxine (TT4) and Vitamin B12. Precision and bias were determined following CLSI EP5 and EP9 guidelines, measured across 3 reagent lots. CLIA 2024 PT criteria provides the allowable total error for the standard Sigma-metric calculation: Sigma-metric = (TEa - |bias|) / SD Performance of the other major analyzers was assessed using the methodology introduced in 2006 by Westgard JO and Westgard SA. The Sigma-metric predicts future problems with PT and potential optimization of QC procedure opportunities, including fewer Westgard Rules, control levels, even reduced QC frequency and materials leading to less cost and time saved. Results Half (50%) of the assays on DxI 9000 achieves 6-Sigma, outperforming all other major manufacturers in the market who range from only 5% to 35% 6-Sigma. Further, over 78% of the DxI 9000 performance was 4Sigma and higher. Less than 8% of performance fell below 3-Sigma, which was observed at the extreme ends of the reportable range. Conclusions The DxI 9000 assay performance is well designed to meet CLIA 2024’s new PT criteria and has the highest prevalence of 6-sigma assay performance observed on market when compared to the top 5 major instrument manufacturers. Assays with Sigma metrics at 4-Sigma and higher are unlikely to face analytical PT difficulties and can be optimized for reduced Westgard Rules, control levels, and potentially even QC frequency.

A-356 A hand-held microfluidic platform for rapid and sensitive identification of respiratory viruses from nasal swab samples

Abstract Background Identifying the infected individual with a fast and accurate test is essential to constrain the spread of viruses and provide the proper treatment. Antigen rapid test (ART) provides quick screening of infection for patients; however, it shows less promising in the detection of early infection and patent’s full recovery as its inferior sensitivity to the nucleic acid detection method. On the other hand, bulky and costly instrument, tedious and prolonged procedures in traditional nucleic acid testing is also unfavorable for the screening. Delta is developing a fully automated hand-held POC using molecular technology to simultaneously detect 4 targets in 20 mins. The POC can be stand-alone or linked to mobile or PC app for different application scenarios. The proprietary recipe of the lysis buffer could neutralize the inhibition in the sample with effective pathogen lysis in seconds. The microfluidic cartridge containing lyophilized reagent can be stored at room temperature. The amplification signal is detected via the optical system from the colorimetric LAMP. This study is to evaluate the performance of the POC system in detecting SARS-CoV2 (SC2), FluA and B viruses in nasal swab. In addition, the assay performance was further compared with other commercial products available. Methods The attenuated SC2 virus, FluA and B viruses were diluted with 1xTE into different concentrations. The diluted viruses were spiked into the lysis buffer in 5% v/v ratio. Nasal samples collected from donors were soaked and swirled inside the cartridges containing lysis buffer according to the manufacturers’ instructions. For commercial products without lysis buffer, direct swabs were used. The cartridges were then put into the respective devices for further testing. Results The lyophilized SC2 assay consistently detected the viruses at 1000 copies/ml (20 out of 20) in real nasal samples in the presence of Delta Lysis Buffer (containing lyophilized neutralizer). However, without Lysis buffer, there was no signal detected even in the presence of 240x LoD viruses. The whole workflow sensitivity was tested with SC2 positive samples at 1x, 4x, 10x LoD and negative samples in the nasal sample matrix on Delta hand-held system. The results were processed via embedded algorithm and displayed on device as well as on PC and mobile app respectively. Out of 79 positive samples tested, the detection rate was 100%. There was no false positive detection for 31 negative samples tested. Positive internal control indicated the valid runs. 5 major SC2 subtypes (99.8% in silico analysis of 366,707 strains), 10 FluA and 9 FluB subtypes were successfully detected. Further, Delta hand-held platform outperformed in the LoD of SC2 with 500 copies/swab comparing to the ≥50,000 copies/swab of the benchmarked commercial platforms. Conclusions The performance of the hand-held molecular detection system we developed demonstrated a rapid, sensitive, and competitive analytical performance in the respiratory panel detection. The capability of device linking to apps and clouds for data management and the extendable docking system for multiple devices to run samples simultaneously and independently, make it applicable for the infectious diseases screening in both clinical settings and for home-use.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R &amp; D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

A-132 Next generation ammonia reagent with improved reagent composition and assay performance

Abstract Background Ammonia is produced by catabolism of protein and other compounds which contain nitrogen. Elevated ammonia levels are toxic for the central nervous system. High plasma ammonia is a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in urea cycle metabolism. The reliable and accurate quantification of ammonia is influenced by assay, reagent, and sample management. Current commercially available GLDH enzyme-based ammonia assay reagents experience high bias between matched Lithium Heparin and K2-EDTA samples, poor accuracy, imprecision at the low end, large HIL (hemolysis, icterus and lipemia) interferences and shorter shelf life. To improve upon the listed deficiencies, we developed an improved GLDH based ammonia assay in a two-part format with improved reagent composition. Methods Ammonia reagent in 2-part format R1 and R2 were assessed for accuracy, precision, interference by HIL reportable range, sensitivity (LoB, LoD, LoQ), shelf life, matched matrix comparison (Lithium Heparin vs K2-EDTA) and method comparison. All testing were performed using Li Heparin plasma samples in Beckman DxC700AU analyzer. Accuracy was tested using Verichem ammonia standards which are ACS ammonia standard material. Twenty-day precision studies were performed using three levels of ammonia concentrations using Bio-Rad Liquichek Ammonia controls and plasma samples. Interference was tested in two ammonia concentrations (40-50 µmol/L) and (200-250 µmol/L) using commercially available spiking material for clinical chemistry assays such as conjugated and unconjugated bilirubin, Intralipid and in-house prepared hemolysate. Infinity Ammonia Reagent on Beckman Coulter AU Chemistry Analyzers was used for method comparison studies Results Next generation ammonia showed good accuracy with Verichem ammonia standards and acceptable within-run and total precision. Studies showed significant improvement in sensitivity and longer shelf life (&amp;gt;18 months). Next generation ammonia is less affected by interferences from icterus (100 mg/dL), lipemia (1000 mg/dL intralipid) and hemolysis (100 mg/dL). Comparison studies show no significant bias between match Lithium Heparin and K2-EDTA matched samples. Conclusions Next Generation Ammonia reagent showed very good overall performance required for reliable and accurate quantification of ammonia in human plasma sample.